Cultures for production of avermectin aglycones

ABSTRACT

Mutants of Streptomyces avermitilis lacking ability to produce glycosylated avermectins and lacking branched-chain 2-oxo acid dehydrogenase activity, method for preparation thereof, and use thereof to produce natural and non-natural avermectin aglycones useful as parasiticides.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a continuation of application Ser. No. 08/060,942, filed May 11,1993, now abandoned, which is a continuation of application Ser. No.07/660,972, filed on Feb. 26, 1991, now U.S. Pat. No. 5,240,850, whichis a continuation of application Ser. No. 07/112,972, filed on Oct. 23,1987, now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to mutants of Streptomyces avermitilis lackingthe ability to produce glycosylated avermectins and lackingbranched-chain 2-oxo acid dehydrogenase activity, to methods forproducing said S. avermitilis and to their use for the production ofnatural and non-natural avermectin aglycones.

2. Description of the Prior Art

U.S. Pat. No. 4,310,519 and 4,429,042 describe the avermectins, acomplex of related agents having potent antiparasitic activity, andtheir production by aerobic fermentation of strains of Streptomycesavermitilis; namely, S. avermitilis ATCC Nos. 31267, 31271 and 31272.The last two strains cited represent a frozen vial and a lyophilizedtube, respectively of a culture obtained by ultraviolet irradiation ofS. avermitilis ATCC 31267.

EP 214,731, published March 18, 1987, the counterpart of U.S. patentapplication Ser. No. 886,867, filed Jul. 16, 1986, discloses a number ofcompounds (referred to herein as non-natural avermectins) related to thenatural or known avermectins but having a novel substituent group at the25-position, and a process for their preparation by fermentation of anavermectin producing organism in the presence of certain specifiedcarboxylic acids, or derivatives or precursors thereof. Also disclosedare the aglycones of said avermectins and their preparation by mild acidhydrolysis of the non-natural avermectins. The S. avermitilis organismsused to produce the said novel C-25 substituted avermectins are S.avermitilis ATCC 31267, 31271, 31272 and NCIB 12121. The latterorganism, derived from S. avermitilis ATCC 31271, gives improved yieldsof the novel C-25 substituted avermectins when it is cultured in asemi-defined medium. Each of ATCC 31267, 31271, 31272 and NCIB 12121 mayalso produce, in addition to the novel C-25 substituted derivative,varying amounts of the known, or natural, avermectins wherein the25-substituent is isopropyl or (S)-sec-butyl (1-methylpropyl).

The carbon skeleton of the avermectins (depicted in formula (I) below)is derived from acetates and propionates and the C-25 substituent ofnatural avermectins from L-isoleucine (R=(S)-sec-butyl) or L-valine(R=isopropyl) [Fisher and Mrozik, "Macrolide Antibiotics", AcademicPress (1984) Ch. 14].

By "known" or "natural" avermectins is meant those avermectins producedby S. avermitilis ATCC 31267, ATCC 31271 and ATCC 31272 wherein the25-position substituent is isopropyl or (S)-sec-butyl(1-methylpropyl).Avermectins wherein the 25-position substituent is other than isopropylor sec-butyl (S-form) are referred to herein as novel or non-naturalavermectins.

The strains of S. avermitilis cited in the above-mentioned patentsproduce a class of substances described generically therein as C-076.The class comprises eight distinct but closely related compoundsdescribed as C-076 A1a, A1b, A2a, A2b, B1a, B1b, B2a and B2b. The "a"series of compounds refers to the natural avermectin wherein the25-substituent is (S)-sec-butyl and the "b" series to those wherein the25-substituent is isopropyl. The designations "A" and "B" refer toavermectins wherein the 5-substituent is methoxy or hydroxy,respectively. Lastly, the numeral "1" refers to avermectins wherein adouble bond is present at the 22-23 position; and numeral "2" toavermectins having a hydrogen at the 22-position and hydroxy at the 23position.

In this application, the "a" and "b" identifiers have been dropped.Identifiers A1, A2, B1 and B2 have been retained to refer to non-naturalavermectins having the structural features corresponding to those of thenatural avermectins as noted above.

Generation of mutants devoid of branched-chain 2-oxo acid dehydrogenaseactivity has been reported for Bacillus subtilis, Willecke and Pardee,J. Biol. Chem. 246, 5264-72 (1971) and Pseudomonas putida, Martin etal., J. Bacteriology, 115 198-204 (1973), but not for Sterptomyces.

U.S. Pat. No. 4,206,205 describes monosaccharide and aglyconederivatives of C-076; i.e., C-076 derivatives in which one or both ofthe carbohydrate moieties of the disaccharide function4-(alpha-L-oleandrosyl) alpha-L-oleandrose attached to C-13 of themacrolide ring has been removed by hydrolysis in an aqueousnon-nucleophilic organic solvent in the presence of an acid, preferablysulfuric acid.

S. avermitilis Agly-1, a mutant strain which produces virtually onlyavermectin aglycones A1a and A2a is reported by Schulman et al. J.Antibiot. 38(11), 1494-1498 (1985). Also reported is the fermentation ofS. avermitilis Agly-1 in the presence of sinefungin which causedincreased production of avermectin aglycone B components. Likewise, S.avermitilis O8, a high producing strain for avermectins, when fermentedin the presence of sinefungin as inhibitor of O-methyl transferases,resulted in production of avermectins lacking O-methyl groups on theaglycone at C-5 and in the oleandrose disaccharide moiety.

U.S. Pat. No. 4,378,353 describes C-076 related compounds and theirpreparation by cultivation of MA-5218, a mutant strain of S. avermitilisATCC 31272, obtained therefrom by ultraviolet irradiation. The mutant isidentified as ATCC 31780. The C-076 related compounds produced by saidmutant have major structural differences from the C-076 compounds. Allthe products lack the C-076 furan ring. Additionally, in certain of thecompounds reported, one or both of the oleandrose sugar moieties havebeen cleaved while in others the 5-position group was oxidized to a ketogroup.

Three classes of O-methyltransferase mutants of S. avermitilis thatproduce avermectins lacking O-methyl groups have been reported by Rubyet al., 6th International Symposium on the "Biology of Actinomycetes",Debrecen, Hungary, Aug. 26-30 (1985) and by Schulman et al,Antimicrobial Agents and Chemotherapy 31, 744-7 (1987). The first classproduces primarily B avermectins due to their inability to methylate theC-5 hydroxyl of the macrocyclic lactone ring. The second class produces3'-O, 3"-O-bis-demethylavermectins (avermectins lacking the O-methylsubstituent at the 3 position of both oleandrose monosaccharideresidues), and which are referred to as demethylavermectins. The thirdclass is unable to methylate at any position.

Schulman et al., Fed. Proc. 44, 931 (1985) disclose increased productionof B avermectins by fermenting S. avermitilis in the presence ofsubstances such as sinefungin, S-adenosylethionine andS-adenosylhomocysteine which inhibit the methylation of the C-5 hydroxygroup of the aglycone moiety by the enzyme avermectinB-O-methyltransferase. Streptomyces avermitilis mutants which lackO-methyltransferase activity and produce increased amounts of avermectinB components are also disclosed and referred to by Schulman et al. inAntimicrobial Agents and Chemotherapy 29, 620-624 (1986).

It has now been found that mutagenesis of S. avermitilis mutants whichlack branched-chain 2-oxo acid dehydrogenase activity produces mutantswhich, when cultivated in an appropriate medium, provide avermectinaglycones. The mutants do not possess the ability to produce naturalavermectin aglycones in the absence of added compound RCOOH wherein R isisopropyl or (S)-sec-butyl, or of a compound convertible to RCOOH duringthe fermentation process. Surprisingly and unexpectedly, however, themutants have been found to produce avermectin aglycones, natural andnon-natural, when fermented in the presence of an added compound R--COOHwherein R is isopropyl or (S)-sec-butyl, or other group disclosedherein, or of a precursor to said RCOOH. It is even more surprising thatthe herein described mutants, which are unable to degrade L-isoleucineor L-valine, are able to assimilate a wide variety of compounds into theavermectin biosynthetic pathway with production of non-naturalavermectin aglycones free of the presence of natural avermectinaglycones.

Certain of the natural avermectin aglycones, A1a and A2a, are producedby S. avermitilis Aglyl-1 as noted above. However, they, along with theaglycones of the remaining natural avermectins are normally prepared byacid hydrolysis of the corresponding avermectin. This procedurenecessitates isolation of the natural avermectins from theirfermentation broths. While the natural avermectins have been isolated insubstantially pure form (see U.S. Pat. No. 4,429,042) the methodologyis, at best, laborious. The overall production of avermectin aglyconesby this procedure is, therefore, even more laborious by reason of theadded step of hydrolysis. The ability to choose to produce eithernatural or non-natural avermectin aglycones so as to minimize the numberand complexity of the products, and by so doing to increase the purityof a chosen avermectin aglycone, and thereby to simplify separationprocedures, is a desirable goal.

SUMMARY OF THE INVENTION

S. avermitilis strains lacking branched-chain 2-oxo acid dehydrogenaseactivity and capable of producing aglycones when fermented in a suitablenutrient medium are obtained by mutation of S. avermitilis strainslacking branched-chain 2-oxo acid dehydrogenase activity. The hereindescribed mutants of this invention are unable to synthesize the naturalavermectin aglycones except where the fatty acid, or a precursorthereto, bearing the isopropyl or sec-butyl (S-form) group is added tothe medium in which the mutants are fermented. They are capable ofproducing natural and non-natural avermectin aglycones when fermentedunder aqueous aerobic conditions in a nutrient medium containing anappropriate primer acid or compound convertible thereto in thefermentation process. The requisite branched-chain 2-oxo aciddehydrogenase deficient strains, e.g.S. avermitilis I-3 (ATCC 53567) areproduced by mutation of avermectin producing strains of S. avermitilisand especially by mutation of S. avermitilis ATCC 31267, ATCC 31271,ATCC 31272 or NCIB 12121.

The mutants, characterized by their lack of branched-chain 2-oxo aciddehydrogenase activity, are selected from amongst the mutagenizedcolonies on the basis of a ¹⁴ CO₂ assay. In this procedure the absenceof ¹⁴ CO₂ evolution by a permeabilized colony from a substrate of [¹⁴C-1]-2-oxoisocaproic acid or [¹⁴ C-1]-2-oxo-3-methylvaleric acid or [¹⁴C-1]-2-oxo-3-methylbutyric acid indicates absence of branched-chain2-oxo acid dehydrogenase activity. The thus-produced mutants are thensubjected to a second mutation. They are then cultivated in anappropriate medium in the presence of an appropriate primer acid and thefermentation products checked by thin-layer chromatography (TLC) and/orhigh performance liquid chromatography (HPLC) for aglycones.Alternatively, as those skilled in the art will recognize, the doublyblocked mutants can be produced in the reverse order; i.e.; thebranched-chain 2-oxo acid dehydrogenase block can be introduced as thesecond rather than the first step.

It was surprising and unexpected that the herein-described mutantslacking branched-chain 2-oxo acid dehydrogenase activity exhibited theability to produce avermectin aglycones, especially non-naturalavermectin aglycones. The inability of the mutants of this invention toproduce the natural fatty acylcoenzyme A derivatives when grown on aconventional medium could have been a lethal mutation if membraneintegrity depended upon said derivatives or if 2-oxo acid accumulationby the mutant led to cytotoxicity. Furthermore, the mutants would nothave been expected to synthesize acetyl-CoA and propionyl-CoA fromL-isoleucine and L-valine degradative metabolism as this requires theenzyme activity that the mutants are missing. The requirement for theseacyl-CoA derivatives for avermectin biosynthesis, noted above, led tothe expectation that the mutants might be severely impaired innon-natural avermectin aglycone production, which, surprisingly, was notthe case.

The lack of 2-oxo acid dehydrogenase activity in the mutants describedherein results in the prevention of branched-chain fatty acyl-CoAsynthesis from the degradation of L-isoleucine and L-valine and,thereby, the synthesis of the natural avermectins. In like manner, it isexpected that branched-chain amino acid transaminase-negative mutants ofS. avermitilis would reduce and possibly prevent the ability to producethe natural avermectins. Such transaminase-negative mutants would not beable to synthesize branched-chain 2-oxo acids from isoleucine and valinevia the normal route of transamination. The reduced availability ofthese 2-oxo acids, which are substrates for the active branched-chain2-oxo acid dehydrogenase enzyme, can effectively prevent branched-chainfatty acyl CoA synthesis. Thus, the present invention also encompassesthe use of such transaminase-negative mutants alone, and mutants inwhich both the branched-chain transaminase negative and 2-oxo aciddehydrogenase-negative mutations are combined.

The present invention also includes any organism, regardless of itsappearance or physiological behavior, that may be developed by means oftransformation, transduction, genetic recombination or some othergenetical procedure, using a nucleic acid or an equivalent material fromthe herein described species, whereby it has acquired thecharacteristics of the herein described mutants.

The terms "avermectin" or "avermectins" as used herein refers tocompounds having formula (I) below but wherein the 25-substituent (R)can be any group assimilable at said position by the S. avermitilis ofthis invention. Avermectin aglycones refers to formula (II) compoundswherein the disaccharide ether moiety,4-(alpha-L-oleandrosyl)-alpha-L-oleandrosyloxy, at C-13 is replaced byOH.

The herein described mutants are highly valuable for producingnon-natural avermectin aglycones by the processes disclosed andexemplified herein. They are especially valuable for production ofpreferred avermectin aglycones, i.e., formula (II) compounds wherein theC-25 substituent is C₄ -C₆ cycloalkyl or cycloalkenyl, optionallysubstituted by C₁ -C₄ alkyl group; 1-methylthioethyl, or a 5- or6-membered oxygen or sulfur heterocyclic group, especially 3-thienyl or3-furyl.

DETAILED DESCRIPTION OF THE INVENTION

Mutation of an avermectin-producing member of the species Streptomycesavermitilis is carried out according to known procedures using any of avariety of mutating agents including ultraviolet irradiation, X-rayirradiation, N-methyl-N'-nitro-N-nitrosoguanidine, ethylmethanesulfonate, nitrous acid and nitrogen mustards, e.g.,N-methylbis(2-chloroethyl)amine, or like treatments. The mutagenesis canbe conducted on spores or on a vegetative culture of S. avermitiliscapable of producing natural avermectins, e.g., S. avermitilis ATCC31272.

Following procedures well known to those skilled in the art, mutagenizedcolonies are selected on the basis of a biochemical assay method whichpermits screening of large numbers of randomly mutagenized bacterialcolonies for ¹⁴ CO₂ production from [¹⁴ C-1]-2-oxo acids (Tabor et al.,J. Bact. 128, 485-486, 1976).

The methodology comprises growing the mutant colonies in the wells of amicrotiter plate on a suitable nutrient medium, permeabilizing the cellswith toluene followed by adding the [¹⁴ C-1]-2-oxo acid (e.g.2-oxoisocaproic acid) to each well and checking the atmosphere above thefermentation for ¹⁴ CO₂. Alternatively, [¹⁴ C-1]-2-oxo-3-methylvalericacid, or [¹⁴ C-1]-2-oxo-3-methylbutyric acid can be used in place [¹⁴C-1]-2-oxo-isocaproic acid. Production of ¹⁴ CO₂ is conveniently checkedfor by placing moist Ba(OH)₂ -- saturated filter paper above theindividual wells to trap any ¹⁴ CO₂ released and detection of Ba¹⁴ CO₃,if any, by autoradiography. Mutants which lack branched-chain 2-oxo aciddehydrogenase activity give autoradiograms approximating those of blankcontrols; i.e., no Ba¹⁴ CO₃ is produced by the mutants

The thus-obtained mutants are subjected to further mutagenesis using anyof the above-mentioned mutating agents. Mutagenized colonies areselected for their ability to produce avermectin aglycones whencultivated in a suitable medium.

The morphological and cultural characteristics of the mutants of thisinvention are generally as described in U.S. Pat. No. 4,429,042, butwith certain exceptions. The distinguishing characteristics of themutants of this invention are their lack of branched-chain 2-oxo aciddehydrogenase activity, and their ability to produce avermectinaglycones when cultivated in a suitable medium as described herein.These characteristics result in the failure of the mutants to producethe natural avermectin aglycones when grown on a defined mediumsubstantially free of fatty acids RCOOH wherein R is isopropyl or(S)-sec-butyl, or compounds convertible to said RCOOH duringfermentation. A taxonomic investigation conducted by the American TypeCulture Collection, confirmed that the characteristics of S. avermitilisI-3, the parental strain, itself a mutant (selected by the above ¹⁴ CO₂assay), bears a close relationship to those of the grandparental ATCC31272 strain described in U.S. Pat. No. 4,429,042. However, mutantstrain I-3 (ATCC 53567) forms significantly fewer spore chains than doesATCC 31272; whilst, in contrast to the description given by Merck forATCC 31272 in U.S. Pat. No. 4,429,042, we are unable to detect growth ofthe mutants or of ATCC 31272 with sucrose as sole carbon source.

Mutant I-3 is deficient only in branched-chain 2-oxo acid decarboxylaseactivity. The doubly-blocked mutant S-2805, obtained by further mutationof I-3 and selected for its ability to produce avermectin aglyconesbears a taxonomic relation to ATCC 31272, similar to that of mutantstrain I-3.

Streptomyces avermitilis I-3, and S-2805 have been deposited under theterms of the Budapest Treaty in the American Type Culture Collection,Rockville, Md., a recognized depository affording permanence of thedeposits and ready accessibility thereto by the public if a patent isgranted on this application. They have been given the designationStreptomyces avermitilis ATCC 53567 and ATCC 53677, respectively. Thedeposits are available during pendency of this application to onedetermined by the Commissioner of the United States Patent and TrademarkOffice to be entitled thereto under 37 CFR 1.14 and 35 USC 122, and inaccordance with foreign patent laws in countries wherein counterparts ofthis application, or its progeny, are filed. All restrictions on theavailability to the public of the microorganisms deposited will beirrevocably removed upon granting of the patent.

Each of S. avermitilis ATCC 31267, ATCC 31271, ATCC 31272 and NCIB 12121produces the natural avermectins, formula (I) compounds ##STR1## whereinthe broken line at the 22-23 position represents an optional doublebond;

R¹ is hydroxy and is present only when the double bond is absent;

R² is 4'-(alpha-L-oleandrosyl)-alpha-L-oleandrosyloxy of the formula##STR2## R³ is hydrogen or methyl; and R is isopropyl or (S)-sec-butyl.U.S. Pat. No. 4,285,963 describes an avermectin of formula (I) whereinthe 25-position is substituted by a methyl and an ethyl group R¹ ishydroxy and R³ is methyl.

In the non-natural avermectins referred to herein R is a substituentother than isopropyl or (S)-sec-butyl and is as defined below.

The mutants of this invention produce avermectin aglycones, formula(II); i.e. compounds of formula (I) but wherein R² is hydroxy. The valueof R in the avermectin aglycones produced by the mutants of thisinvention can correspond to the group (isopropyl or (S)-sec-butyl)present in the natural avermectins, or to a group R wherein R is agroup, other than isopropyl or (S)-sec-butyl, assimilable at the25-position by the herein-described mutants.

The compounds L-valine and L-isoleucine essential for the biosynthesisof natural avermectins and their aglycones [formulae (I) and (II)] occurin the cell of S. avermitilis. These compounds are believed to enterinto the biosynthesis of avermectins via conversion to 2-oxo acid anddecarboxylation of the acid by branched-chain 2-oxo acid dehydrogenase,concomitant with coupling the product with coenzyme A. Their presenceaccounts for the concurrent production of both the isopropyl and(S)-sec-butyl compounds of formulae (I) and (II). This, of course, givesrise to problems in separating the isopropyl from the (S)-sec-butylderivatives.

When fermented in a nutrient medium containing the appropriate primercompound the mutants of this invention produce a compound of formula(II) or, as is more usually the case, a mixture of two or more compoundsof formula (II) in which R corresponds to the primer compound used. Upto four products, conveniently and trivially referred to as R-avermectinA1 aglycone, A2 aglycone, B1 aglycone and B2 aglycone, can be produced.The "R-" group, of course, refers to the C-25 substituent. For example,when R is cyclopentyl the four possible avermectin aglycones are:

    ______________________________________                                        Trivial Name     R.sup.1       R.sup.3                                        ______________________________________                                        cyclopentyl      double bond   CH.sub.3                                       avermectin A1                                                                 aglycone                                                                      cyclopentyl      hydroxy       CH.sub.3                                       avermectin A2                                                                 aglycone                                                                      cyclopentyl      double bond   H                                              avermectin B1                                                                 aglycone                                                                      cyclopentyl      hydroxy       H.                                             avermectin B2                                                                 aglycone                                                                      ______________________________________                                    

In the non-natural avermectin aglycone the C-25 substituent "R" is otherthan isopropyl or (S)-sec-butyl.

Compounds of formula (II) wherein the double bond is present and OH isabsent may alternatively be prepared from the corresponding compound offormula (II) wherein R¹ is OH and the double bond is absent by adehydration reaction. The reaction is performed by first selectivelyprotecting the hydroxy groups at the 5 and 13 positions, e.g. as thet-butyldimethylsilyloxy acetyl derivative, then reacting with asubstituted thiocarbonyl halide, such as (4-methylphenoxy)thiocarbonylchloride, followed by heating in a high boiling point solvent, e.g.trichlorobenzene, to effect the dehydration according to proceduresdescribed in U.S. Pat. No. 4,328,335. The product is finally deprotectedto give the unsaturated compound.

Formula (II) compounds wherein R³ is H may also be prepared from thecorresponding compounds wherein R³ CH₃ by demethylation. This reactionis achieved by treating the 5-methoxy compound, or a suitably protectedderivative thereof, with mercuric acetate and hydrolyzing the resulting3-acetoxy enol ether with dilute acid to give the 5-keto compound. Thisis then reduced using, for example, sodium borohydride to yield the5-hydroxy derivative. Appropriate reagents and reaction conditions forthese steps are described in U.S. Pat. No. 4,423,209.

Compounds of formula (II) wherein R¹ is H and the double bond is absentcan be prepared from the corresponding compound wherein the double bondis present and R¹ is absent, by selective catalytic hydrogenation usingan appropriate catalyst. For example, the reduction may be achievedusing tris(triphenylphosphine)rhodium (I) chloride as described inEuropean Patent Application Publication No. 0001689.

The aglycones, formula (II), can also be prepared from the correspondingformula (I) compounds (R² is4'-(alpha-L-oleandrosyl)-alpha-L-oleandrosyloxy) by removing the4'-(alpha-L-oleandrosyl)-alpha-L-oleandrose group by mild hydrolysiswith an acid in an aqueous organic solvent to yield the aglycone havinga hydroxy group at the 13-position.

The compounds capable of utilization by the S. avermitilis of thisinvention for the biosynthesis of avermectins, natural and non-natural,are compounds of formula (III-A)

    R--COOH                                                    (III-A),

including compounds convertible to (III-A) during the fermentationprocess. Said compounds are referred to herein as "primer compounds". Informula (III-A), R is an alpha-branched-chain group, the carbon atomthereof to which is attached the --COOH group is also attached to atleast two other atoms or groups other than hydrogen. This definition, ofcourse, embraces saturated and unsaturated acyclic and cyclic groups,including those optionally bearing a sulfur or oxygen heteroatom as amember of the acyclic chain or cyclic ring.

More specifically, R, which becomes the C-25 substituent, can be analpha-branched C₃ -C₈ alkyl, alkenyl, alkynyl, alkoxyalkyl oralkylthioalkyl group; a C₅ -C₈ cycloalkylalkyl group wherein the alkylgroup is an alpha-branched C₂ -C₅ alkyl group; a C₃ -C₈ cycloalkyl or C₅-C₈ cycloalkenyl group, either of which may optionally be substituted bymethylene or one or more C₁ -C₄ alkyl groups or halo atoms (fluoro,chloro, iodo or bromo); or a 3 to 6 membered oxygen or sulfur containingheterocyclic ring which may be saturated, or fully or partiallyunsaturated and which may optionally be substituted by one or more C₁-C₄ alkyl groups or halo atoms.

Compounds convertible to RCOOH (III-A); i.e., precursors, in thefermentation process are compounds of formulae (III-B) wherein R is asdefined above:

    R--(CH.sub.2).sub.n --Z                                    (III-B)

n is 0, 2, 4 or 6; and Z is --CH₂ OH, --CHO, --CH₂ NH₂, --COOR⁵ or--CONHR⁶ wherein R⁵ is H or (C₁₋₆)alkyl; R⁶ is hydrogen, (C₁₋₄)alkyl, orthe residue of an amino acid, especially of aspartic acid, glutamic acidand methionine, e.g., --CH(COOH)CH₂ COOH, --CH(COOH) (CH₂)₂ COOH and--CH(COOH) (CH₂)₂ SCH₃, respectively.

Also included in this invention are the isomeric forms of formula(III-A) and (III-B) compounds, and compounds convertible thereto duringthe fermentation process, and the isomeric avermectin aglycones at C-25resulting from their use in the herein described process.

The process of this invention is carried out by aerobic fermentationwith a strain of S. avermitilis which lacks ability to produceglycosylated avermectins and branched-chain 2-oxo acid dehydrogenaseactivity in an aqueous nutrient medium comprising an assimilable sourceof nitrogen, carbon, inorganic salts and a compound of formula RCOOH, ora compound convertible to said compound (i.e., a precursor) during thefermentation. The acid, or compound convertible thereto, is added to thefermentation either at the time of inoculation or at intervals duringthe fermentation. Production of the avermectin aglycone products may bemonitored by removing samples from the fermentation, extracting with anorganic solvent and following the appearance of the product bychromatography, for example using high performance liquidchromatography. Incubation is continued until the yield of the producthas been maximized, generally for a period of from 4 to 15 days.

A preferred level of each addition of the primer compounds (carboxylicacid or compound convertible thereto) is between 0.05 and 3.0 grams perliter. The primer compound can be added continuously, intermittently orall at once to the fermentation. The acid (RCOOH) is added as such or asa salt, such as the sodium, lithium or ammonium salt, or as a compoundconvertible to the acid as defined above. The acid, if a solid, ispreferably dissolved in a suitable solvent such as water or(C₁₋₄)alcohols.

The media used for the fermentation can, especially when the C-25substituent is to be isopropyl or (S)-sec-butyl, be conventional mediacontaining assimilable sources of carbon, nitrogen and trace elements.When the C-25 substituent is to be a non-natural group; i.e., it is notisopropyl or (S)-sec-butyl, the fermentation medium is one in which thechosen ingredients lack, or contain only minimal amounts of primercompounds wherein the R moiety is isopropyl or (S)-sec-butyl.

After fermentation for a period of several days at a temperaturepreferably in the range of 24° to 33° C., the fermentation broth iscentrifuged or filtered and the mycelial cake is extracted withpreferably acetone or methanol. The solvent extract is concentrated andthe desired product is then extracted into a water-immiscible organicsolvent, such as methylene chloride, ethyl acetate, chloroform, butanolor methyl isobutyl ketone. The solvent extract is concentrated and thecrude product is further purified as necessary by chromatography, forexample using preparative reverse phase, high performance liquidchromatography.

The product is generally obtained as a mixture of the compounds offormula (II) wherein R¹ is OH and the double bond absent or R¹ is absentand the double bond is present and wherein R³ is H or CH₃ ; however, theproportions can vary depending on the particular mutant and primercompound employed and the conditions used.

The source of the R group; i.e., whether it comes directly from R--COOHor is produced from one of the above precursors, or from any precursor,is immaterial to the production of the avermectin aglycones. Thecritical requirement of the process of this invention for theirproduction is that the desired R group be made available to the S.avermitilis strains of this invention in the fermentation process.

Suitable compounds include the following:

2,3-dimethylbutyric acid

2-methylhexanoic acid

2-methylpent-4-enoic acid

2-cyclopropyl propionic acid

4,4-difluorocyclohexane carboxylic acid Lithium salt

4-methylenecyclohexane carboxylic acid

3-methylcyclohexane carboxylic acid (cis/trans)

1-cyclopentene carboxylic acid

1-cyclohexene carboxylic acid

tetrahydropyran-4-carboxylic acid

thiophene-2-carboxylic acid

3-furoic acid

2-chorothiophene-4-carboxylic acid

cyclobutane carboxylic acid

cyclopentane carboxylic acid

cyclohexane carboxylic acid

cycloheptane carboxylic acid

2-methylcyclopropane carboxylic acid

3-cyclohexene-1-carboxylic acid

2-methylthiopropionic acid

2-methyl-4-methoxybutyric acid

thiophene-3-carboxylic acid

hydroxymethylcyclopentane

3-thiophene carboxaldehyde

3-cyclohexylpropionic acid

3-cyclopentylpropionic acid

hydroxymethylcyclobutane

tetrahydrothiophene-3-carboxylic acid

3-cyclopentyl-1-propanol

3-methylcyclobutane carboxylic acid Lithium salt

3-fluorocyclobutane carboxylic acid

3-methylenecyclobutane carboxylic acid Lithium salt

2-methyl-4-methylthiobutyric acid

tetrahydrothiopyran-4-carboxylic acid

cyclobutylmethylamine

ethyl cyclobutanecarboxylate

4-hydroxymethylcyclopentene

2-(3-thiophenecarbonyl)propionic acid ethyl ester

S-2-methylpentanoic acid

R-2-methylpentanoic acid

O-methyltransferase mutants can be obtained from the herein-describedbranched-chain 2-oxo acid dehydrogenase negative, aglycone-producingmutants. Mutations in active branched-chain 2-oxo acid dehydrogenaseactivity, combined with an O-methyltransferase mutation, yield strainsof S. avermitilis that will, when fed RCOOH compounds or compoundsconvertible to RCOOH during the fermentation process, produce primarilyB avermectins. Said mutants are obtained by mutagenesis of the hereindescribed mutants which lack branched-chain 2-oxo acid dehydrogenaseactivity by means of ultraviolet light and/or chemical mutagens such asN-methyl-N-nitrosourethan, nitrosoguanidine or other agent such as thoseenumerated above. Alternatively, branched-chain 2-oxo acid dehydrogenasepositive mutants which lack the O-methyltransferase can be mutated bytreatment with UV light or a mutagenizing agent to produce thebranched-chain 2-oxo acid dehydrogenase negative, aglycone-producingmutants.

In addition to production of desired alleles of a given strain ofmicroorganism by mutagenesis, protoplast fusion permits introduction ofdesirable alleles produced/identified in one strain into the chromosomeof another strain. For example, a strain of S. avermitilis deficient inbranched-chain 2-oxo acid dehydrogenase activity and the ability toglycosylate avermectins can, by protoplast fusion with a5-O-methyltransferase deficient S. avermitilis strain, produce a strainof S. avermitilis capable of synthesizing only avermectin B aglycones.

The non-natural avermectin aglycones produced by such mutants arecharacterized by the presence of hydroxy groups at the C-5 position ofthe aglycone moiety.

The above-described mutants are identified according to the methodologydescribed by Schulman et al., Antimicrobial Agents and Chemotherapy, 29,620-624 (1986). They are useful for the same purposes and in the sameway as are the known avermectin aglycones.

Alternatively, increased amounts of the B avermectins are produced byfermenting the mutants of this invention, which lack activebranched-chain 2-oxo acid dehydrogenase, in the presence of a substancesuch as sinefungin, S-adenosylethionine or S-adensylhomocysteine whichinhibits O-methyl transferase activity.

The compounds of the invention are highly active antiparasitic agentshaving particular utility as anthelmintics, ectoparasiticides,insecticides and acaricides.

Thus the compounds are effective in controlling; i.e., preventing andtreating, a variety of conditions caused by endoparasites including, inparticular, helminthiasis which is most frequently caused by a group ofparasitic worms described as nematodes and which can cause severeeconomic losses in swine, sheep, horses and cattle as well as affectingdomestic animals and poultry. The compounds are also effective againstother nematodes which affect various species of mammals (humans andanimals) including, for example, Dirofilaria in dogs and variousparasites which can infect humans including gastro-intestinal parasitessuch as Ancylostoma, Necator, Ascaris, Strongyloides, Trinchinella,Capillaria, Trichuris, Enterobius and parasites which are found in theblood or other tissues and organs such as filiarial worms and theextract intestinal states of Stronqyloides and Trichinella.

The compounds are also of value in treating ectoparasite infectionsincluding in particular arthropod ectoparasites of animals and birdssuch as ticks, mites, lice, fleas, blowfly, biting insects and migratingdipterous larvae which can affect cattle and horses.

The compounds are also insecticides active against household pests suchas the cockroach, clothes moth, carpet beetle and the housefly as wellas being useful against insect pests of stored grain and of agriculturalplants such as spider mites, aphids, caterpillars and against migratoryorthopterans such as locusts.

The compounds of formula (II) are administered as a formulationappropriate to the specific use envisaged and to the particular speciesof host animal being treated and the parasite or insect involved. Foruse as an anthelmintic the compounds may be administered orally in theform of a capsule, bolus, tablet or a liquid drench, or alternatively,they may be administered by injection or as an implant. Suchformulations are prepared in a conventional manner in accordance withstandard veterinary practice. Thus capsules, boluses or tablets may beprepared by mixing the active ingredient with a suitable finely divideddiluent or carrier additionally containing a disintegrating agent and/orbinder such as starch, lactose, talc, magnesium stearate etc. A drenchformulation may be prepared by dispersing the active ingredient in anaqueous solution together with dispersing or wetting agents, etc., andinjectable formulations may be prepared in the form of a sterilesolution which may contain other substances, for example, enough saltsor glucose to make the solution isotonic with blood. These formulationswill vary with regard to the weight of active compound depending on thespecies of host animal to be treated, the severity and type of infectionand the body weight of the host. Generally for oral administration adose of from about 0.02 to 10 mg per kg of animal body weight given as asingle dose or in divided doses for a period of from 1 to 5 days will besatisfactory, but, of course, there can be instances where higher orlower dosage ranges are indicated and such are within the scope of thisinvention.

As an alternative the compounds may be administered with the animalfeedstuff and for this purpose a concentrated feed additive or premixmay be prepared for mixing with the normal animal feed.

For use as an insecticide and for treating agricultural pests thecompounds are applied as sprays, dusts, emulsions and the like inaccordance with standard agricultural practice.

Production of S. avermitilis I-3 (ATCC 53567)

Step 1. S. avermitilis ATCC 31272 was grown as a confluent lawn on NewPatch Agar Medium for 12 days at 30° C. The medium comprised

    ______________________________________                                        V-8 Juice*                 200    ml                                          CaCO.sub.3                 3      grams                                       Agar                       15     grams                                       H.sub.2 O          to      1000   ml                                          Nutrient broth             1.0    grams/L                                     sodium acetate.3H.sub.2 O  1.4    grams/L                                     isovaleric acid            50     mg/L                                        isobutyric acid            50     mg/L                                        2-methylbutyric acid       50     mg/L                                        isoleucine                 250    mg/L                                        leucine                    250    mg/L                                        valine                     250    mg/L                                        trace elements solution**  1      ml/L                                        ______________________________________                                        *A mixture of 8 vegetable juices (tomato, carrots, celery, beets,             parsley, lettuce, watercress and spinach) plus salt, ascorbic and             citric acids and natural flavors. Available from Campbell Soup                Company, Camden, NJ.                                                          **Composition of Trace elements solution:                                     FeCl.sub.3.6H.sub.2 O                                                                    2.7 g                                                              MnSO.sub.4.H.sub.2 O                                                                     4.2                                                                CuSO.sub.4.5H.sub.2 O                                                                    0.5                                                                CaCl.sub.2 11.0                                                               H.sub.3 BO.sub.3                                                                         0.62                                                               CoCl.sub.2.6H.sub.2 O                                                                    0.24                                                               ZnCl.sub.2 0.68                                                               Na.sub.2 MoO.sub.4                                                                       0.24                                                           

Dissolve the above in 1 liter of 0.1N HCl.

Spores were harvested from 3 such plates and suspended in 20 ml. of0.05M tris-maleic acid buffer, pH 9.0.

Step 2. 10 ml of the spore suspension was added to a vial containing 10mg of N-methyl-N'-nitro-N-nitrosoguanidine (NTG). The vial was incubatedand shaken at 28° C. for 60 minutes and the spores then washed profuselywith 1% NaCl solution.

Step 3. The washed spores were suspended in 1% NaCl and mixed with anequal volume of 80% ethylene glycol. This suspension was preserved at-20° C. and used as a source of cells to be screened for mutants. Itgave approximately 10⁴ colonies/ml when germinated.

This spore stock was spread on YPD plates to yield approximately 100colonies per plate (YPD medium comprises 10 g/l of each of yeastextract, Bacto peptone* and dextrose; and 15 g/l of Bacto agar*,adjusted to pH 6.9 before autoclaving) Ingredients marked with anasterisk are available from Difco Laboratories, Detroit, Mich. 48238.

step 4. Single colonies were picked from plates after 2-3 weeks ofgrowth at 28° C. and placed in individual wells of a standard 96 wellmicrotiter plate. Also, a small quantity of the colony was patched ontoa fresh agar medium to serve as a source of viable cells when mutantsare identified.

Step 5. To each well was added approximately 75 microliters of a liquidM9 salts medium containing 1% glucose, 0.1% casamino acids, and 0.01% ofeach of isovaleric, isobutyric and 2-methylbutyric acids. After severaldays of incubation at 28° C., the cells were assayed for the presence ofbranched-chain 2-oxo acid dehydrogenase. (Each liter of M9 salts mediumcomprises 6 g Na₂ HPO₄, 3 g KH₂ PO₄, 0.5 g NaCl and 1 g of NH₄ Cl. Themedium is autoclaved and then 1 ml of each of sterilized 1M MgSO₄ and0.1M CaCl₂ are added aseptically).

Step 6. A microsuspension of 5% toluene in M9 salts medium was preparedby a brief sonication of the immiscible mixture. To 25 ml of thissuspension was added 1.2 ml of a solution containing [¹⁴C-1]-2-oxo-isocaproic acid, 2.5 microcurie/ml and 10.0microcurie/micromole. 50 Microliters of this overall mixture was addedto each of the wells of the microtiter plates containing the colonies tobe assayed.

Step 7. The ¹⁴ CO₂ produced from each well was trapped and visualized bythe procedure described by Tabor et al., J. Bacteriol. 128 485-486(1976) entitled "Convenient Method for Detecting ¹⁴ CO₂ in MultipleSamples: Application to Rapid Screening for Mutants". Mutants lackingactive branched-chain 2-oxo acid dehydrogenase produce no Ba¹⁴ CO₃beyond that observed for the controls.

A more refined method which improves the contrast between a positiveassay for ¹⁴ CO₂, indicated by a dark spot on the autoradiogram as aresult of Ba¹⁴ CO₃ formation, and a negative assay indicated by no spotor a very light spot, comprises the following modified screen.

Single colonies (see Step 4 above) were picked from the agar mediumafter 7-14 days of growth (rather than 2-3 weeks and assayed directly bysteps 6 and 7 above). Step 5 of the above procedure is omitted.

An even more refined assay method which is quantitative in nature asregards ¹⁴ CO₂ release comprises growing the mutants detected by theabove screens on a suitable medium comprising M9 salts medium withglucose, 1% and "Syncasa-bcaa", 0.1% (a synthetic mixture of L-aminoacids with the approximate composition of commercial casamino acids, butwithout the presence of L-valine, L-isoleucine and L-leucine, seebelow).

    ______________________________________                                        Composition of "Syncasa - bcaa", 100 fold Concentrate                                      grams/liter                                                      ______________________________________                                        L-alanine      3                                                              L-arginine     4                                                              L-aspartic acid                                                                              6                                                              L-cystine      1                                                              L-glutamic acid                                                                              20                                                             glycine        1                                                              L-histidine    2                                                              L-lysine       7                                                              L-methionine   3                                                              L-phenylalanine                                                                              6                                                              L-proline      10                                                             L-serine       6                                                              L-threonine    4                                                              L-tyrosine     4                                                              L-tryptophan   1                                                              ______________________________________                                    

The mixture is adjusted to pH 7 and filter sterilized. One volume ofconcentrate is added to 99 volumes of medium to achieve standard useconcentrations.

After growth to high cell density, the cells were washed in M9 saltsmedium and resuspended in cold M9 salts medium containing 1% toluenewhich had been sonicated to produce a milky white dispersion of thetoluene. The cell/buffer/toluene suspension was incubated for 40 minutesat 30° C. in order permeabilize the cells. The permeabilized cells werethen washed in M9 medium salts and finally resuspended in one-fifth theoriginal volume of M9 medium buffer. 180 Microliters of this suspensionwere used per assay.

A reaction volume of 300 microliters contained the toluenized cells,thiamine pyrophosphate (TPP), 0.4 mM; coenzyme A (CoA), 0.11 mM;nicotinamide adenine dinucleotide (NAD), 0.68 mM, dithiothreitol (DTT),2.6 mM; MgCl₂, 4.1 mM; Tris-HCl, 60 mM; Tris-HCl, 60 mM, pH 7.5; and [¹⁴C-1]-alpha-ketoisocaproate, 6,000 cpm, microcurie per micromole. Theefficiency of counting was 73%. The reaction was carried out in 15 mlscintillation vials containing a 2×2 cm Whatman #4 paper square pressedinto the screw cap of the vial. The paper contains 30 microliters of 1MHyamine Hydroxide (1M solution of methylbenzethonium hydroxide inmethanol; available from Sigma Chemical Co., St. Louis, Mo. 63178),which traps ¹⁴ CO₂ evolved in the reaction. After incubation for 2hours, the papers are immersed in 10 ml of Beckman Aquasol II (UniversalLSC (liquid scintillation counter) available from New England NuclearResearch Products, Boston, Mass. 02118) and the radioactivity measuredin a liquid scintillation counter after equilibration in this solventfor 4 hours or more. A blank control reaction (i.e.--no cells) gives ca.50-300 cpm.

Mutant I-3 and others like it gave counts that were less than or equalto the blank control reaction, whereas the parent strain gave countsseveral fold higher than the blank control value.

Isolation of S. avermitilis S-2805 (ATCC 53677)

Step 1. Approximately 100 mg of S. avermitilis I-3 (ATCC 53567), grownon a fresh SAMM agar plate for four days, was inoculated into a 300 mlflask containing 50 ml of SCM medium (pH 7.2). The flask was then shakenat 200 RPM and 30° C. for twenty-four hours (final pH=8.2).

Step 2. The flask was removed from the shaker and 10 ml of the wholebroth centrifuged in a sterile tube for five minutes at 2000 RPM. Thecells were then resuspended in 50 ml of SCM medium in sterile 300 ml.Erlenmeyer flasks and the flasks shaken on a rotary shaker for two hoursat 30° C.

Step 3. The 10 ml of the suspension was placed in a sterile tube.

Step 4. Ethylmethane sulfonate was added to the tube (in a wellventilated hood), the contents thoroughly mixed, then poured into asterile 300 ml flask and the flask shaken in a rotary shaker for threehours at 30° C.

Step 5. Fresh sterile SCM medium (40 ml) was added to the flask andshaking continued for a total of 70 hours at 30° C.

Step 6. The flask was removed, the contents spun down at 8000 RPM forten minutes at 20° C. The cells were washed by re-suspending in SCMmedium, spun down again and re-suspended in 10 ml SCM medium.

    ______________________________________                                        SCM MEDIUM                                                                    ______________________________________                                        Yeast autolysate        10     g/l                                            Beef extract            5      g/l                                            Casein enzymatic hydrolysate                                                                          10     g/l                                            1M MgSO.sub.4           3      g/l                                            1M K.sub.2 HPO.sub.4 ; pH 7.0 (HCl)                                                                   100    g/l                                            ______________________________________                                    

Step 7. The mutagenized population is diluted and spread for singlecolonies on SAMM agar.

                  SAMM Agar                                                       ______________________________________                                                      g/L                                                             ______________________________________                                        Na.sub.2 HPO.sub.4                                                                            6.0                                                           KH.sub.2 PO.sub.4                                                                             3.0                                                           NaCl            0.5                                                           NH.sub.4 Cl     1.0                                                           1M MgSO.sub.4   1.0                                                           0.1M CaCl.sub.2 1.0                                                           Dextrose        8.0                                                           Casamino Acids  20.0                                                          Agar            20.0                                                          ______________________________________                                    

Step 8. Colonies of the mutagenized population (3% ethylmethanesulfonate) of Streptomyes avermitilis strain I-3 (ATCC 53567) arepicked, and spread as patches on an agar medium prepared as follows(grams per liter): thinned starch, 80; K₂ HPO₄, 1; MgSO₄.7H₂ O, 1;ardamine PH, 5; CaCO₃, 5; P-2000, 1 ml; FeSO₄.7H₂ O, 0.01; MnCl₂.4H₂ O,0.001; ZnSO₄.7H₂ O, 0.001; Bacto agar, 17; distilled H₂ O to 980 ml. ThepH is adjusted to 7.0 with NaOH prior to autoclaving at 121° C. for 20minutes. After autoclaving, 20 ml of a sterile 5% stock solution of(±)-2-methylbutyric acid, pH 7.0 is added.

The agar cultures are incubated 8 to 12 days at 28° C. Cells (mycelia)are removed from the agar surface, and put into 250 microliters ofacetone. Twenty-five (25) microliters of the acetone extracts are thenspotted on Analtech Silica Gel GF precoated thin layer chromatographyplates. The chromatogram is run for 30 to 40 minutes with ethyl acetateas solvent, then dried, and sprayed with 3% vanillin in ethanol. Theplates are placed in a 100° C. oven for 1 to 3 minutes, then sprayedwith 3% sulfuric acid in ethanol, and again placed in a 100° C. oven for10 to 15 minutes. Aglycone-producing cultures are identified by theappearance of a novel spot (Rf ca. 0.63). This migrates coincident to A2aglycone prepared by acid hydrolysis of A2a (1% H₂ SO₄ in methanol, 25°C., 18 hours).

EXAMPLES 1-5

A frozen vial of culture S. avermitilis S-2805 (ATCC 53677) wasinoculated into 100 ml of AS-7 medium in a 500 ml triple-baffled flask.The flask was incubated on a rotary shaker with agitation at 200 rpm at28°-30° C. After 28 hours of incubation, 5 ml of the whole broth wasinoculated into another 100 ml of AS-7 medium in 500 ml triple-baffleflask. The flask was again incubated on a rotary shaker with agitationat 200 rpm, at 28°-30° C. After 24 hours of incubation, 1 ml of thewhole broth was inoculated into 300 ml flasks containing 40 ml of AP-5medium.

Duplicate 40 ml fermentations were run at 28°-30° C. in the presence of440 ppm of each of the primer compounds listed below. The time ofaddition of the primer compounds (RCOOH) to the fermentation is given inthe right hand column.

    ______________________________________                                                            Time of                                                   Primer Compound     Addition                                                  ______________________________________                                        cyclohexane carboxylic acid                                                                       24 hours                                                  cyclopentane carboxylic acid                                                                      96 hours                                                  3-thiophene carboxylic acid                                                                       96 hours                                                  2-methylthiopropionic acid                                                                        24 hours                                                  2-methylbutyric acid                                                                              96 hours                                                  ______________________________________                                    

After 264 hours, the whole broth samples were diluted with water (10mls) and extracted with methylene chloride (2×20 mls). The organicextract was dried (MgSO₄) and evaporated to dryness. The resultingresidues were dissolved in methanol (1 ml) and 30 μl of solutioninjected onto a Beckman Ultrasphere ODS column (3.9 250 mm). The columnwas eluted with a mixture of methanol and 0.1M ammonium acetate (85:15 )at a flowrate of 1 ml per minute. The column effluent was passeddirectly into a VG 12-250 thermospray mass spectrometer.

    __________________________________________________________________________          HPLC          Calcd.                                                    Avermectin                                                                          Ret.          mol.                                                      Aglycone                                                                            Time                                                                              Component Identity                                                                      wt. Characteristic ions (m/e)                             __________________________________________________________________________    sec-butyl                                                                           5.69                                                                              B2a       602 603(MH.sup.+), 602(M.sup.+), 585                                              (MH.sup.+ -H.sub.2 O), 567 (MH.sup.+ -2H.sub.2                                O)                                                    sec-butyl                                                                           6.56                                                                              A2a       616 634(MNH.sub.3.sup.+), 617(MH.sup.+),                                          616(M.sup.+), 599(MH.sup.+ -H.sub.2 O),                                       581(MH.sup.+ -2H.sub.2 O)                             sec-butyl                                                                           8.39                                                                              B1a       584 585(MH.sup.+), 567(MH.sup.+ -H.sub.2 O)               sec-butyl                                                                           10.08                                                                             A1a       598 617(MNH.sub.3.sup.+), 599(MH.sup.+),                                          581(MH.sup.+ -H.sub.2 O)                              cyclopentyl                                                                         7.62                                                                              25-Cyclopentyl A2                                                                       628 646(MNH.sub.3.sup.+), 629(MH.sup.+),                                          628(M.sup.+), 611(MH.sup.+ -H.sub.2 O),                                       593(MH.sup.+ -2H.sub.2 O)                             cyclopentyl                                                                         12.36                                                                             25-Cyclopentyl A1                                                                       610 628(MNH.sub.3.sup.+), 611(MH.sup.+),                                          593(MH.sup.+ -H.sub.2 O)                              3-thienyl                                                                           5.43                                                                              25(3-Thienyl) A2                                                                        642 660(MNH.sub.3.sup.+), 643(MH.sup.+),                                          642(M.sup.+), 625(MH.sup.+ -H.sub.2 O)                3-thienyl                                                                           7.79                                                                              25(3-Thienyl) A1                                                                        624 642(MNH.sub.3.sup.+), 625(MH.sup.+),                                          607(MH.sup.+ -H.sub.2 O)                              __________________________________________________________________________

    ______________________________________                                        AS-7 Medium                                                                                  g/l                                                            ______________________________________                                               thinned starch.sup.a                                                                    20                                                                  Ardamine pH.sup.b                                                                       5                                                                   Pharmamedia.sup.c                                                                       15                                                                  CaCO.sub.3                                                                              2                                                            ______________________________________                                         .sup.a Prepared by hydrolysis of starch by alphaamylase from Bacillus         licheniformis (available from Novo Enzyme, Wilton, CT and sold under the      trademark "Termamyl") to a dextrose equivalent of 40% + 5%                    .sup.b From Yeast Products, Inc., Clifton, NJ 07012                           .sup.c From Traders Protein, Memphis, TN 38108                           

Adjust pH to 7.2 with 25% NaOH.

    ______________________________________                                        AP-5 Medium                                                                                       g/l                                                       ______________________________________                                        thinned starch.sup.a  80                                                      Ardamine pH.sup.b     5                                                       K.sub.2 HPO.sub.4     1                                                       MgSO.sub.4.7H.sub.2 O 1                                                       NaCl                  1                                                       CaCO.sub.3            7                                                       FeSO.sub.4.7H.sub.2 O 0.01                                                    MnCl.sub.2.7H.sub.2 O 0.001                                                   ZnSO.sub.4.7H.sub.2 O 0.001                                                   P-2000 (antifoam)     1      ml/l                                             ______________________________________                                    

Adjust pH to 6.9 with 25% NaOH.

EXAMPLE 6

25-Cyclopentylavermectin A2 aglycone

A frozen inoculum (2 ml) of a culture of Sterptomyces avermitilis mutantorganism ATCC 53677 was inoculated into 50 mls of a medium containingstarch (1 g), Pharmamedia (Trademark) (0.75 g), ardamine pH (0.25 g),and calcium carbonate (0.1 g) in a 300 ml flask and incubated at 28° C.for 2 days. This inoculum (50 ml) was transferred to a second inoculumflask (600 ml) containing starch (12 g), Pharmamedia (8 g), ardamine pH(3 g) and calcium carbonate (1.2 g) and incubated at 28° C. for afurther 2 days. This inoculum was used to inoculate 15 liters of amedium containing starch (1.5 kg), magnesium sulphate (15 g).Pharmamedia (75 g), dipotassium hydrogen phosphate (15 g), ferroussulphate (0.12 g), calcium carbonate (105 g), glutamic acid (9 g, zincsulfate (0.015 g) and manganous sulfate (0.015 g) contained in a 15liter jar fermenter. The fermentation was incubated at 28° C., withagitation at 350 r.p.m. and aeration at 15 liters per minute.Cyclopentane carboxylic acid (6 g) was added after 96 hours and againafter 216 hours (6 g). After 240 hours the mycelium was removed byfiltration and extracted with acetone (5 L +0.75 L wash). The acetoneextract was concentrated to approximately 1.5 L and extracted with ethylacetate (3 L) in two portions. The resulting ethyl acetate layers werecombined and evaporated to give a brown oil (1.2 g).

The above oil was dissolved in diethyl ether and added to a column ofsilica gel (40 g). The column was eluted with diethyl ether and 15 mlfractions were collected. Fractions 4-6 were combined and thenevaporated to yield partially purified material (1.35 mg). The productwas dissolved in methanol (0.5 ml) and chromatographed on a C18 ZorbaxODS (Trademark, Dupont) column (21 mm×25 cm) eluting with a mixture ofmethanol and water (75:25) at a flow rate of 9 mls per minute. Therelevant fractions were combined and the solvent evaporated to yield thecompound of formula (I) wherein R¹ is OH, the double bond is absent, R²is cyclopentyl, R³ is CH₃ and R⁴ is OH as a white powder (9 mg) m.p.131°-133° C.

The structure of the product was confirmed by mass spectrometry asfollows:

Fast atom bombardment mass spectrometry was performed on a VG Model7070E mass spectromer using a sample matrix of triethylene glycol withsolid sodium chloride. (M+Na)⁺ observed at m/e 651 (theoretical 651).

Electron impact mass spectrometry was performed using a VG Model 7070Fmass spectometer. The m/e values for the principal fragments were: 628(M⁺), 610, 468, 335, 317, 275, 251, 233, 223, 179.

We claim:
 1. A biologically pure culture of Streptomyces avermitiliswhich produces only avermectin aglycones having an isopropyl or a(S)-sec-butyl group at C-25 when fermented in an aqueous nutrient mediumcomprising an assimilable source of carbon, nitrogen and inorganic saltsunder aerobic conditions only when said medium further comprises an acidof the formula R--COOH, or a precursor of said acid of the formulaR-(Ch₂)_(n) --Z where n is 0, 2, 4 or 6; Z is --CH₂ OH, --CHO, --COOR⁵,--CH₂ NH₂, or --CONHR⁶ wherein R⁵ is H or (C₁₋₆)alkyl and R⁶ ishydrogen, (C₁₋₄) alkyl, --CH (COOH) CH₂ COOH, --CH (COOH) (CH₂)₂ COOH or--CH (COOH) (CH₂)₂ SCH₃, wherein R is isopropyl or (S) -sec-butyl, saidS. avermitilis lacking branched-chain 2-oxo acid dehydrogenase activitysuch that permeabilized cells thereof are unable to produce ¹⁴ CO₂ fromadded [¹⁴ C-1]-2-oxo-isocaproic acid.
 2. A biologically pure culture ofStreptomyces avermitilis which produces only avermectin aglycones havingan isopropyl or a (S)-sec-butyl moiety at C-25 upon fermentation in anaqueous nutrient medium comprising an assimilable source of carbon,nitrogen and inorganic salts under aerobic conditions only when saidmedium further comprises an acid of the formula R--COOH, or a precursorof said acid of the formula R-(CH₂)_(n) --Z where n is 0, 2, 4 or 6; Zis --CH₂ OH, --CHO, --COOR⁵, --CH₂ NH₂ or --CONHR⁶ wherein R⁵ is H or(C₁₋₆)alkyl and R⁶ is hydrogen, (C₁₋₄)alkyl, --CH(COOH)CH₂ COOH,--CH(COOH) (CH₂)₂ COOH or --CH(COOH) (CH₂)₂ SCH₃ wherein R is isopropylor (S)-sec-butyl.
 3. A strain of Streptomyces avermitilis which producesonly avermectin aglycones having an isopropyl or a (S)-sec-butyl moietyat C-25 wherein (a) said strain is obtained by mutating Streptomycesavermitilis ATCC 53567; and (b) said strain, when fermented in anaqueous nutrient medium comprising an assimilable source of carbon,nitrogen and inorganic salts under aerobic conditions, produces onlysaid avermectin aglycones having an isopropyl or a (S)-sec-butyl moietyat C-25 only when said medium further comprises an acid of the formulaR--COOH, or a precursor of said acid of the formula R-(CH₂)_(n) --Zwhere n is 0, 2, 4 or 6; Z is --CH₂ OH, --CHO, --COOR⁵, --CH₂ NH₂, or--CONHR⁶ wherein R⁵ is H or (C₁₋₆)alkyl and R⁶ is hydrogen, (C₁₋₄)alkyl,--CH(COOH)CH₂ COOH, --CH(COOH) (CH₂)₂ COOH or --CH(COOH)(CH₂)₂ SCH₃wherein R is isopropyl or (S)-sec-butyl.